With one exception all mammalian nitroreductases are markedly inhibited by oxygen. Previous investigations have found that the oxygen-sensitive nitroreductase of rat liver supernatant is DT diaphorase. The reduction of 4-nitroquinoline-1-oxide (4-NQO) by supernatant was inhibited by oxygen only 10%, whereas dicoumarol inhibited 94%. The microsomal reduction of 4-NQO is oxygen sensitive as is characteristic of the usual nitroreductases. Several lines of evidence suggest that the oxygen inhibition of nitroreductases is the result of the air oxidation of the first reduction intermediate, the nitroaromatic anion free radical (RNO-2). RNO2 plus O2 yields RNO2 plus O-2. In view of the known rapid rate of the air oxidation of nitro anion free radical (k is greater than 2.5 x 10 to the fifth power M -1 sec -1), this oxygen-insensitive nitroreductase must not form the RNO-2 or, at the very least, the radical must remain bound to the DT diaphorase and not be accessible to O2. Presumably in this case, the first reduction intermediate formed is the corresponding nitroso compound, the two-electron reduction product.